RESUMEN
The melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic transmembrane receptor involved in pigmentation, adaptive tanning, and melanoma resistance. We report MC1R-mediated or pharmacologically-induced cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435, which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA, and elevates UV-induced mutagenesis. This study mechanistically links cAMP-PKA signaling to NER and illustrates potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals.
Asunto(s)
Daño del ADN , Receptor de Melanocortina Tipo 1/genética , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular Tumoral , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Mutagénesis/efectos de la radiación , Fosforilación/efectos de la radiación , Pigmentación/genética , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/efectos de la radiación , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Rayos Ultravioleta , Proteína de la Xerodermia Pigmentosa del Grupo A/genéticaRESUMEN
People with pale skin, red hair, freckles and an inability to tan--the 'red hair/fair skin' phenotype--are at highest risk of developing melanoma, compared to all other pigmentation types. Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAF(V600E), into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r(e/e) background. Selective absence of pheomelanin synthesis was protective against melanoma development. In addition, normal Mc1r(e/e) mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1r(e/e) mouse skin. These data suggest that the pheomelanin pigment pathway produces ultraviolet-radiation-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage. Although protection from ultraviolet radiation remains important, additional strategies may be required for optimal melanoma prevention.
Asunto(s)
Color del Cabello/genética , Melanoma/genética , Pigmentación de la Piel/genética , Rayos Ultravioleta , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Melaninas/metabolismo , Ratones , Ratones Endogámicos C57BL , Monofenol Monooxigenasa/genética , Peroxidasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Receptor de Melanocortina Tipo 1/genética , Sulfonamidas/farmacología , Análisis de Supervivencia , Células Tumorales CultivadasRESUMEN
Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection (1). Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.
Asunto(s)
Colforsina/administración & dosificación , Glicoproteínas/biosíntesis , Piel/efectos de los fármacos , Piel/metabolismo , Quemadura Solar/metabolismo , Quemadura Solar/prevención & control , Toxinas Biológicas/biosíntesis , Animales , Colforsina/química , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Epidermis/efectos de la radiación , Eritema/etiología , Eritema/prevención & control , Ratones , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Raíces de Plantas/química , Plectranthus/química , Tolerancia a Radiación , Piel/efectos de la radiación , Pigmentación de la Piel/efectos de los fármacos , Rayos UltravioletaRESUMEN
We previously reported that topical application of forskolin to the skin of fair-skinned MC1R-defective mice with epidermal melanocytes resulted in accumulation of eumelanin in the epidermis and was highly protective against UV-mediated cutaneous injury. In this report, we describe the long-term effects of chronic topical forskolin treatment in this animal model. Forskolin-induced eumelanin production persisted through 3 months of daily applications, and forskolin-induced eumelanin remained protective against UV damage as assessed by minimal erythematous dose (MED). No obvious toxic changes were noted in the skin or overall health of animals exposed to prolonged forskolin therapy. Body weights were maintained throughout the course of topical forskolin application. Topical application of forskolin was associated with an increase in the number of melanocytes in the epidermis and thickening of the epidermis due, at least in part, to an accumulation of nucleated keratinocytes. Together, these data suggest in this animal model, short-term topical regular application of forskolin promotes eumelanin induction and that over time, topical forskolin treatment is associated with persistent melanization, epidermal cell accumulation, and skin thickening.
Asunto(s)
Colforsina/administración & dosificación , Colforsina/farmacología , Protectores contra Radiación/farmacología , Pigmentación de la Piel/efectos de los fármacos , Pigmentación de la Piel/efectos de la radiación , Baño de Sol , Rayos Ultravioleta , Administración Tópica , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/efectos de la radiación , Colforsina/efectos adversos , Hígado/anatomía & histología , Hígado/efectos de los fármacos , Hígado/efectos de la radiación , Melaninas/biosíntesis , Melaninas/metabolismo , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Melanocitos/efectos de la radiación , Ratones , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/efectos de la radiación , Receptor de Melanocortina Tipo 1/metabolismo , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Fenómenos Fisiológicos de la Piel/efectos de la radiación , Factores de TiempoRESUMEN
The K14-SCF transgenic murine model of variant pigmentation is based on epidermal expression of stem cell factor (SCF) on the C57BL/6J background. In this system, constitutive expression of SCF by epidermal keratinocytes results in retention of melanocytes in the interfollicular basal layer and pigmentation of the epidermis itself. Here, we extend this animal model by developing a compound mutant transgenic amelanotic animal defective at both the melanocortin 1 receptor (Mc1r) and tyrosinase (Tyr) loci. In the presence of K14-Scf, tyrosinase-mutant animals (previously thought incapable of synthesizing melanin) exhibited progressive robust epidermal pigmentation with age in the ears and tails. Furthermore, K14-SCF Tyr(c2j/c2j) animals demonstrated tyrosinase expression and enzymatic activity, suggesting that the c2j Tyr defect can be rescued in part by SCF in the ears and tail. Lastly, UV sensitivity of K14-Scf congenic animals depended mainly on the amount of eumelanin present in the skin. These findings suggest that c-kit signaling can overcome the c2j Tyr mutation in the ears and tails of aging animals and that UV resistance depends on accumulation of epidermal eumelanin.